JWA Antagonizes Paraquat-induced Neurotoxicity Via Activation Of Nrf2

Parkinson’s disease(PD)is a progressive neurodegenerative disease that is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta(SNpc).Recent advances have revealed that both genetic and environmental factors contribute to the initiation and development of PD,however,the underlying molecular mechanisms have not been fully elucidated.Thus,understanding the mechanisms of the gene-environment interaction during the onset and progression of PD is urgent for the prevention and treatment of PDParaquat(PQ,1,1’-dimethyl-4,4’-bipiridinium),a widely used pesticide in agriculture,has been regarded as one of major risk factors for PD.In addition,the chemical structure of PQ is similar to that of MPTP(1-methyl-4-phenyl-1,2,3,6-tetrahydropiridiium),a widely used toxicant for the generation of PD mouse models.PQ generates superoxide anion radical(O2·)in the central nervous system,and triggers a series of reactions to produce excess reactive oxygen species(ROS),which permanently damage neurons.Furthermore,ROS accumulation disrupts the homeostasis of intracellular glutathione and activities of several antioxidant enzymes.Nuclear factor erythroid-2 related factor(Nrf2),a key factor involved in the response to oxidative stress,modulates expression of antioxidant proteins and detoxifying enzymes by binding to the antioxidant response element.Under oxidative stress,Nrf2 is phosphorylated by a variety of protein kinases(MAPKs,PKC and PI3K),and is subsequently translocated into the nucleus to activate expressions of target genes.The JWA gene,which is initially identified as an all-trans retinoic acid-responsive factor,repairs oxidative stress-triggered DNA damage by regulating the base excision repair pathway.Moreover,our previous study has demonstrated that astrocytic JWA protects DA neurons from MPTP-induced degeneration by inhibiting NF-κB activity and ROS production.However,whether JWA has a similar protective effect on PQ-induced damage in neurons is still unknown.In this study,two neuron cell lines(HT-22 and SH-SY5Y)and neuron-specific JWA knockout(JWA-nKO)and age-matched wild-type(JWA-nWT)mice were exposed to PQ.The results indicated that JWA protects neurons from PQ-induced damage and apoptosis by up-regulating Nrf2.Objective:To investigate the effect of JWA on PQ-induced damage and apoptosis in neuron cells and to explore the underlying mechanisms of how JWA regulates Nrf2 to antagonize the neurotoxicity induced by PQ exposure.Methods:The neuron-specific JWA knockout(JWA-nKO)and age-matched wild-type(JWA-nWT)mice were used for the chronic PQ mouse model.Establishment of the chronic PQ mouse model:JWA-nKO and JWA-nWT mice received an intraperitoneal injection of 7 mg/kg PQ every 2 days;the mice received 8 injections in total.After finishing the model,behavioral tests,including the rotarod test,pole test and open-field test,were performed.Mice were sacrificed one week after the final injection,protein samples from the SNpc were extracted and slices were prepared for detecting the TH expression in the midbrain.PQ-induced damage,apoptosis and oxidative stress indicators in HT-22 and SH-SY5Y cells transfected with Flag-JWA,siRNA JWA and the control plasmids were determined by Western blot,TUNEL assay and DCFH-DA assay.Appropriate commercial kits were used for detecting the contents of GSH and GPx.Relative antioxidant enzymes were detected by PCR and the key molecule for antagonizing neurotoxicicity induced by PQ.Furthermore,how JWA regulates the relative biomarker,Nrf2 was determined by Western blot and PCR.Finally,the expression of Nrf2 in JWA-nKO and JWA-nWT mice midbraines was determined by immunohistochemistry.Result:1.Neuron-specific JWA knockout mice(JWA-nKO)are vulnerable to paraquat.PQ treatment led to significant weight loss in JWA-nKO mice starting on the 8th day after exposure;at the end of the trial,the weight of JWA-nKO mice in PQ group was about 84.80%compared with that at the start of the trial.In the rotarod test,the latency to fall from the rotarod in JWA-nKO mice was 116.63 ± 19.18 s,which was significantly decreased compared with JWA-nWT mice(218.63 ± 39.84 s)(P<0.05).Similarly,in the pole test,the descending time(T-TLA)of JWA-nKO mice was 34.00±13.18 s,which was significantly delayed compared with that of JWA-nWT mice after PQ treatment(17.89 ± 6.84 s).In addition,Nissl staining revealed that JWA-nKO mice mice lost more neurons in the SNpc than JWA-nWT mice,but this difference was more pronounced after PQ treatment.2.Neuron cell lines are sensitive to PQ treatment.CCK-8 assay showed that the 24 h IC50 to PQ in somatic cell line 293T cells was 3698 μM,and that in HT-22 cells was 258 μM,and 450 μM in SH-SY5Y cells.In addition,the 48 h IC50 to PQ in HT-22 was 80 μM and that in SH-SY5Y cells was 157 μM.3.PQ treatment mediates JWA upregulation in damaged and apoptotic neuron cell lines.The expressions of apoptotic markers(cleaved Caspase-3 and cleaved PARP-1)showed a time-dependent upregulation pattern under PQ treatment,after 36 h treatment of PQ,the expressions of apoptotic markers increased to 3-4 folds compared with the basic levels,which was accompanied by gradually increased JWA expression.DNA damage markers(XRCC1,polβ and p53)were increased in a concentration-dependent manner under PQ treatment,which correlated with the elevation level of JWA.4.JWA ameliorates PQ induced DNA damage in neuron cell lines.HT-22 and SH-SY5Y cells with higher JWA expression improved survival rates.Immunofluorescence assay revealed that the expression of 8-OHdG negatively correlated with the level of JWA expression.Determinations of SOD and MDA demonstrated that the SOD level in JWA knock-down neuron cells decreased to 89.32%and 92.00%,respectively,MDA level increased to 1.45 and 1.61 folds,respectively.5.JWA attenuates PQ induced neuronal apoptosis by reducing reactive oxygen species(ROS)production.Upregulation of cleaved PARP-1 and Caspase-3 were negatively correlated with the level of JWA,and these trends were enhanced by PQ Accordingly,TUNEL-positive cell numbers were negatively associated with JWA levels.The TUNEL-positive cell numbers in JWA knock-down cells was 1.36 folds compared with the control group,and in JWA overexpressing cells,it accounted for 69.13%compared with the control group.Further investigations revealed that ROS production was also negatively correlated with JWA levels in HT-22 and SH-SY5Y cells.6.JWA increases intracellular GSH and GPx levels and transcriptional Nrf2 expression under PQ treatment.JWA upregulated the levels of GPx and GSH in both HT-22 and SH-SY5Y cells.In HT-22 and SH-SY5Y cells overexpressed JWA,levels of GPx increased to 1.60 and 1.13 folds,respectively,levels of GSH incrased to 1.30 and 1.50 folds.Here,qPCR analysis revealed that the expressions of Nrf2,GPx and HO-1 were significantly decreased in JWA knock-down cells,and markedly increased in JWA overexpressing cells.7.JWA increases Nrf2 expression through the PI3K/AKT and MAPK/ERK cascades.The levels of p-ERK,p-AKT(Thr308)and Nrf2 were regulated by JWA expression.In addition,pharmacological inhibition of MEK/ERK by U0126 or PI3K/AKT by LY294002 abolished the Nrf2 experession and protective effect of JWA against PQ treatment.The cell survival rates in JWA overexpressing cells with pharmacological inhibitions decreased to 90.20%and 81.63%compared with the control group.Moreover,elevated activities of SOD,MDA,GSH and GPx induced by JWA were also compromised by inhibiting the MEK/ERK or PI3K/AKT8.JWA downregulates the neuronal expression levels of tyrosine hydroxylase and Nrf2 in mice.The protein levels of TH and Nrf2 were significantly decreased in JWA-nKO mice treated with PQ compared with those in JWA-nWT mice.The Nrf2 expression in JWA-nKO mice was 14.33%compared with JWA-nWT mice,and TH expression was 21.96%compared with JWA-nWT mice.However,there was no significant difference between the two genotypes of mice treated with saline.Accordingly,immunohistochemistry confirmed that the level of Nrf2 was markedly decreased in JWA-nKO mice after PQ treatment.Conclusion:Our findings uncover that JWA protects neurons from PQ damage through regulating MAPK/ERK-Nrf2 and PI3K/AKT-Nrf2 signaling pathways.JWA is a key molecule for regulating the neuronal damage and apoptosis induced by PQ,and it may be a novel target for prevention and treatment of PQ-induced neurotoxicity and related diseases.